ABSTRACT Malaria caused half a million deaths and affected more than 200 million people worldwide in 2016. The disease is caused by Plasmodium parasites transmitted by the bites of infected mosquitoes. Thousands of sporozoites are often present in the salivary glands of mosquitoes but only few of these are injected during a mosquito bite. Our understanding of the regulation of this critical parasite stage has been hampered by the difficulty to obtain sufficient amount of study material and, in particular, the reliance on pools of parasites has precluded examining variations among sporozoites. Here, we propose to use single-cell RNA sequencing to characterize the gene expression regulation of individual sporozoites and to rigorously assess gene expression heterogeneity among them. We will first study P. falciparum sporozoites and assess the effect of several key parameters on sporozoite gene expression. Second, we will compare the profiles of sporozoites from Plasmodium species with and without liver dormancy to test the hypothesis that the development into hypnozoites is pre-programmed in sporozoites. Overall, our studies will provide unique insights on the biological processes underlying the maturation and fate of Plasmodium sporozoites, and will significantly improve our understanding of malaria transmission as well as provide a solid foundation for developing better malaria vaccines and drugs targeting the eluding dormant parasites.